Article Comprising Calcium for Reducing the Production of TSST-1

ABSTRACT

Articles comprising one or more calcium salts are provided. The articles can contain one or more calcium salts in an amount effective to reduce the production of TSST-1 by at least about 50% when measured by the Shake Flask Method. In certain embodiments, the one or more calcium salts can be substantially non-lethal to  Staphylococcus aureus  when measured by the Shake Flask Method, and/or to  Lactobacillus crispatus, Lactobacillus gasseri , and/or  Lactobacillus iners  when measured by the Maximum Tolerated Dose Test.

FIELD OF THE INVENTION

The present invention relates generally to articles including calcium,more particularly to articles including calcium for use in and aroundthe human vagina.

BACKGROUND OF THE INVENTION

In females between the age of menarche and menopause, the normal vaginaprovides an ecosystem for a variety of microorganisms that is typicallymaintained in a relatively delicate balance. Bacteria are thepredominate type of microorganisms present in the vagina, and most womenharbor about 10⁷ to 10⁹ colony forming units (CFU) per ml of vaginalsecretion. The more commonly isolated bacteria include lactic acidbacteria, Lactobacillus species, Corynebacteria species, Gardnerellavaginalis, Staphylococcus species, Peptococcus species, aerobic andanaerobic streptococcal species, Bacteroides species and Prevotellaspecies. Other microorganisms that have been isolated from the vagina onoccasion include yeast (Candida albicans), protozoa (Trichomonasvaginalis), mycoplasma (Mycoplasma hominis), chlamydia (Chlamydiatrachomatis), and viruses (Herpes simplex). These latter organisms aregenerally associated with vaginitis or sexually transmitted diseases,although they may be present in low numbers without causing symptoms.

Physiological, social and idiosyncratic factors can affect the celldensity and species of microbes present in the vagina. Physiologicalfactors can include age, day of the menstrual cycle, and pregnancy.Social and idiosyncratic factors can include presence and method ofbirth control, sexual practices, systemic disease (e.g., diabetes), andmedication. Disruption of the vaginal ecosystem, such as, e.g., normalhealthy vaginal microflora, can permit opportunistic infections toemerge.

Toxic shock syndrome (“TSS”) is characterized by rapid onset of highfever, vomiting, diarrhea and rash followed by a drop in blood pressureand vital organ failure. The causative agent of toxic shock syndrome isthought to be exotoxin-producing cocci, such as, e.g., Staphylococcus,e.g., S. aureus, and/or Streptococcus, e.g., S. pyogenes. The exotoxinsassociated with TSS can include, for example, Staphylococcus:Enterotoxin A, Enterotoxin B, Enterotoxin C, and Toxic Shock SyndromeToxin-1 (TSST-1), and Streptococcus: pyrogenic Exotoxin A, Exotoxin B,Exotoxin C. It is believed that TSS is not caused by the presence of thebacteria per se, but rather by the toxic effects of the associatedexotoxin.

TSS has been associated with the use of absorbent articles within thevagina. The syndrome has also been observed with surgical dressings andnasal packing. TSS appears to occur with elevated frequency inassociation with absorbent pads having high levels of absorbency.

Various modifications to the absorbent articles have been proposed toreduce the risk of TSS associated with absorbent articles. Typically,such modifications can adversely affect levels of S. aureus, or otherbacteria that make up the vaginal microflora, such as, e.g., byemploying antimicrobials or bactericidal agents. These effects can upsetthe healthy balance discussed above. In addition, modifications to theabsorbent article may not be compatible with manufacture and/or storagetechniques and/or may degrade over time.

Accordingly, it would be desirable to provide an improved article,including an absorbent article suitable for use in and around themammalian vagina, which reduces or prevents the production of TSST-1.

SUMMARY OF THE INVENTION

Articles comprising one or more calcium salts are provided. The articlescan contain one or more calcium salts in an amount effective to reducethe production of TSST-1 by at least about 50% when measured by theShake Flask Method. In certain embodiments, the one or more calciumsalts can be substantially non-lethal to Staphylococcus aureus whenmeasured by the Shake Flask Method. In certain embodiments, the one ormore calcium salts can be substantially non-lethal to Lactobacilluscrispatus, Lactobacillus gasseri, and/or Lactobacillus iners whenmeasured by the Maximum Tolerated Dose Test.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to articles comprising calcium salts orcompounds. In certain embodiments, the article can include one or morecalcium salts in an amount effective to reduce the production of TSST-1.In addition, or alternatively, the one or more calcium salts can besubstantially non-lethal to normal vaginal microflora, such as, e.g.,Staphylococcus and Lactobacillus species. Thus, the calcium salts canreduce the production of TSST-1 while generally not affecting normalvaginal microflora, such as bacteria including, e.g., Staphylococcusaureus, Lactobacillus crispatus, Lactobacillus gasseri, and/orLactobacillus iners.

As used herein, the term “article” refers to any article wherein thereduction or prevention of the generation of toxins from Gram positivebacteria would be beneficial. Suitable articles can include, e.g.,sanitary napkins, panty liners, adult incontinent undergarments,diapers, medical bandages, absorbent articles intended for medical,dental, surgical and/or nasal use, and/or non-absorbent articlesintended for use in the human vagina, such as, e.g., pessaries andfemale contraceptive devices. In certain embodiments, the article can bean absorbent article and/or a vaginal article.

As used herein, the term “absorbent article” refers to devices thatabsorb and/or contain a substance, such as, e.g., body exudates. Atypical absorbent article can be placed against or in proximity to thebody of the wearer to absorb and contain various body exudates.Absorbent articles can include, e.g., tampons, surgical wound dressings,sponges, and nasal packings.

As used herein, the term “vaginal article” includes articles intended tobe worn in or near the vagina, such as, e.g., disposable absorbentarticles that can be worn by women for menstrual and/or lightincontinence control, such as, for example, sanitary napkins, tampons,interlabial products, incontinence articles, and liners. Non-absorbentproducts intended to be worn in the vagina such as, e.g., pessaries forthe treatment of vaginal prolapse and/or incontinence, cervical caps,contraceptive sponges, menstrual cups, and contraceptive diaphragms arealso included.

As used herein, the term “tampon” refers to any type of absorbentstructure such as, e.g., an absorbent mass, that can be inserted intothe vaginal canal or other body cavity, such as, e.g., for theabsorption of fluid therefrom, to aid in wound healing, and/or for thedelivery of materials, such as moisture or active materials such asmedicaments. In general, the term “tampon” is used to refer to afinished tampon after the compression and/or shaping process.

As used herein, the term “pledget” refers to an absorbent material priorto the compression and/or shaping of the material into a tampon.Pledgets are sometimes referred to as tampon blanks or softwinds.

As used herein, the term “vaginal canal” refers to the internalgenitalia of the human female in the pudendal region of the body. Theterms “vaginal canal” or “within the vagina” as used herein are intendedto refer to the space located between the introitus of the vagina andthe cervix.

As used herein the term “non-lethal” with regard to bacteria means thecell density of the bacteria is not reduced by more than a factor ofabout 10 CFU/ml (1 log) of test fluid relative to the control test fluidas measured by the Maximum Tolerated Dose Test (“MTDT”) forLactobacillus species and as measured by the Shake Flask Method for S.aureus.

As used herein the term “lethal” with regard to bacteria means the celldensity of the bacteria are reduced by at least a factor of about 10³CFU/ml (3 log) of test fluid relative to the control test fluid asmeasured by the Maximum Tolerated Dose Test for Lactobacillus speciesand as measured by the Shake Flask Method for S. aureus.

As used herein, the term “stable” means the calcium salt can have TSST-1reducing capability as measured by the Shake Flask Method when exposedto conditions such as, e.g., during manufacture and/or storage.

As used herein the term “fugitive” means the calcium salt is capable ofmoving through the fiber matrix of an article, such as an absorbentarticle, when the article comprises a plurality of fibers.

As used herein, the term “bound” means less than about 10% of thecalcium added to the absorbent article is removed by soaking the articleover an 8 hour period at 100° F. in three times the syngyna capacity ofsterile physiologic saline solution. The syngyna capacity is determinedby the syngyna test (U.S. FDA 21 CFR 801.430, Revised as of Apr. 1,2006). The percent of “bound” calcium is calculated as (calcium presentin the article−calcium in solution) divided by calcium present in thearticle.

As used herein, the term “partially bound” means less than about 50% ofthe calcium added to the absorbent article is removed by soaking thearticle over an 8 hour period at 100° F. in three times the syngynacapacity of sterile physiologic saline solution.

As used herein, the term “substantially bound” means less than about 25%of the calcium added to the absorbent article is removed by soaking thearticle over an 8 hour period at 100° F. in three times the syngynacapacity of sterile physiologic saline solution.

In certain embodiments, an article can include one or more calcium saltsin an amount effective to reduce the production of TSST-1. Theproduction of TSST-1 can be reduced by any suitable amount in the ShakeFlask Method, such as, e.g., about 50%, about 60%, about 70%, about 80%,about 90%, about 95%, or more. In certain embodiments, the amount ofTSST-1 can be measured by the Shake Flask Method, described below.

In addition, or alternatively, the one or more calcium salts can besubstantially non-lethal to Staphylococcus aureus, such as, e.g., whenmeasured by the Shake Flask Method, and/or can be substantiallynon-lethal to Lactobacillus crispatus, Lactobacillus gasseri, and/orLactobacillus iners, such as, e.g., when measured by the MaximumTolerated Dose Test. As such, in certain embodiments, S. aureus, L.crispatus, L. gasseri, and/or L. iners are not reduced by more than afactor of about 10 CFU/ml (1 log) of test fluid relative to the controltest fluid as measured by the Maximum Tolerated Dose Test, such as,e.g., a factor of about 9 CFU/ml of test fluid, a factor of about 8CFU/ml of test fluid, a factor of about 7 CFU/ml of test fluid, a factorof about 6 CFU/ml of test fluid, a factor of about 5 CFU/ml of testfluid, a factor of about 4 CFU/ml of test fluid, a factor of about 3CFU/ml of test fluid, a factor of about 2 CFU/ml of test fluid, or less.

In certain embodiments, the calcium salt can have a solubility greaterthan about 0.3 millimoles/L of water at 25° C., such as, e.g., greaterthan about 1 millimoles/L of water, greater than about 2 millimoles/L ofwater, greater than about 4 millimoles/L of water, greater than about 6millimoles/L of water, greater than about 8 millimoles/L of water,greater than about 10 millimoles/L of water at 25° C., or more.

The calcium salt can be compatible with the tampon making and/or storageprocess. For example, in certain embodiments, the calcium salt canretain its ability to inhibit toxin following the making process and/orwhen stored in conditions, such as, e.g., high and/or low temperatures,related to the commercial manufacture and sale. In certain embodiments,the calcium salt can retain its ability to inhibit toxin for anysuitable time at any suitable temperature, such as, e.g., at about 100degrees Celsius for about 3 hours, from about −30 degrees Celsius toabout 65 degrees Celsius for about 24 hours, from about 0 degreesCelsius to about 50 degrees Celsius, or any other suitable temperature.

In certain embodiments, the calcium salt can resist acquisition ofmoisture in a humid environment. Articles, such as, e.g., tampons and/orother absorbent articles, can be exposed to conditions where therelative humidity can exceed 80%, such as, e.g., during commercialstorage and/or as in the bathroom during a shower. Some salts, such as,e.g., calcium chloride, magnesium chloride, and/or zinc chloride, aredeliquescent substances that can have a strong affinity for moisture andcan absorb relatively large amount of fluid from the atmosphere. The useof a deliquescent substance in a tampon can result in moisture beingdrawn into the tampon such that the tampon can expand, causing insertioninto the body or expulsion from the applicator to be difficult. Incertain embodiments, the calcium salt can absorb less than about 50% ofits initial dry weight as determined by drying 3 hours at 100 degrees C.and then exposed to 80% relative humidity (RH) at 23 degrees C. for 22hours, such as, e.g., less than about 45%, less than about 40%, lessthan about 35%, less than about 30%, less than about 25%, less thanabout 20%, less than about 15%, or less of its initial dry weight.

In certain embodiments, the one or more calcium salts can be calciumlactate and/or calcium citrate malate.

The article can be any suitable article where the reduction of TSST-1toxin is desirable and/or beneficial. Suitable articles include, e.g.,sanitary napkins, tampons, panty liners, interlabial products, adultincontinent undergarments, diapers, surgical wound dressings, sponges,nasal packings, other absorbent articles intended for medical, dental,surgical and/or nasal use, and/or non-absorbent articles intended foruse in the human vagina, such as, e.g., pessaries for the treatment ofvaginal prolapse and/or incontinence, cervical caps, contraceptivesponges, menstrual cups, and contraceptive diaphragms. In certainembodiments, the article can be suitable for use with a mammal, such as,e.g., a human.

In certain embodiments, the article can be a tampon. The tampon can beformed from a pledget that can be constructed from a wide variety ofliquid-absorbing materials suitable for use in absorbent articles. Suchmaterials include, for example, rayon (such as GALAXY rayon (a tri-lobedrayon) or DANUFIL rayon (a round rayon), both available from KelheimFibres GmbH of Kelheim, Germany), cotton, folded tissues, wovenmaterials, nonwoven webs, synthetic and/or natural fibers or sheeting,comminuted wood pulp, which is generally referred to as airfelt, foams,or combinations of these materials. Examples of other suitable materialsinclude: creped cellulose wadding; meltblown polymers including coform;chemically stiffened, modified or cross-linked cellulosic fibers;synthetic fibers such as crimped polyester fibers; peat moss; foam;tissue including tissue wraps and tissue laminates; or any equivalentmaterial or combinations of materials, or mixtures of these.Additionally, superabsorbent materials, such as superabsorbent polymersor absorbent gelling materials can be incorporated into the tampon.

The tampon can include one or more withdrawal cords and/or overwraps.The withdrawal cord and/or overwrap can be any suitable material, suchas, for example, rayon, cotton, bicomponent fibers, polyethylene,polypropylene, other suitable natural or synthetic fibers known in theart, and mixtures thereof. In certain embodiments, the tampon cancomprise an overwrap material that substantially encloses the compressedtampon. The tampon can also or alternatively include a secondaryabsorbent member, such as, for example, a mass of secondary absorbentmaterial attached to the withdrawal cord proximate the withdrawal end ofthe tampon. Suitable secondary absorbent members are described in, e.g.,U.S. Pat. No. 6,258,075.

Any suitable amount of calcium salt can be added and/or included in thearticle. Suitable amounts include, e.g., an amount effective to reducethe production of TSST-1. In certain embodiments, the amount of calciumadded to the article can be greater than about 0.009 millimoles, greaterthan about 0.01 millimoles, greater than about 0.02 millimoles, greaterthan about 0.04 millimoles, greater than about 0.06 millimoles, greaterthan about 0.08 millimoles, greater than about 0.1 millimoles, greaterthan about 0.5 millimoles, greater than about 1 millimoles, greater thanabout 2 millimoles, greater than about 3 millimoles, greater than about4 millimoles, greater than about 5 millimoles, greater than about 6millimoles, greater than about 7 millimoles, greater than about 8millimoles, greater than about 8 millimoles, greater than about 9millimoles, greater than about 10 millimoles or more. In certainembodiments, less than substantially all of the calcium salt that isadded to the article is available, such as, e.g., when some of thecalcium salt is retained within the article during use and/or when lessthan substantially all of the calcium salt can be recovered from thearticle after addition.

The calcium salt can be added to an article by any suitable processand/or at any step in the manufacturing process. In certain embodiments,such as, e.g., when adding calcium salt to a tampon, the calcium saltcan be added to the absorbent fiber during the process prior to makingthe pledget, for example, in the fiber washing and drying steps and/orwhen a fiber finishing agent is added to facilitate fiber processing.Alternatively, or in addition, the salt can be added to the fiber beforethe pledget is made or after the pledget is made as an aqueous solutionor suspension, or in a non-aqueous solution or suspension or even as apowder. For example, calcium can be added to one or more layers of apledget prior to compression by exposing one or more portions of thepledget to an aqueous solution or suspension containing the calcium.Examples of methods for exposing a tampon pledget to an aqueous solutioninclude, e.g., spraying the aqueous solution on the pledget, dipping thepledget in the aqueous solution and/or washing the pledget with theaqueous solution. Alternatively, or in addition, one or more calciumsalts can be incorporated in the tampon after compression, such as, forexample by exposing a substantially completed tampon to an aqueoussolution containing the calcium and then drying the tampon. Optionally,the calcium can employ one or more pharmaceutically acceptable andcompatible carrier materials. Some suitable examples of carriermaterials include, e.g., aqueous solutions, gels, foams, lotions, balms,salves, ointments, boluses, suppositories, and/or combinations thereof.In certain embodiments, it is also possible to add the calcium salt as apowder when the pledget or article is manufactured.

The calcium salt can be included in one or more portions of an article.One such example can be a tampon having the calcium salt incorporatedinto or on the primary absorbent member, the overwrap, the secondaryabsorbent member and/or the withdrawal means. In certain embodiments,the calcium salts can also be distributed on, within and/or throughoutone or more portions of the tampon. The calcium salt can also beincorporated directly into the absorbent fiber or into the fibercomprising the overwrap during manufacturing of the fiber. In certainembodiments, such as, e.g., when using polyethylene fibers,polypropylene fibers, polyethylene terephthalate fibers, conjugatefibers, bicomponent fibers, rayon fibers, and/or any other suitablesynthetic fibers, the calcium salt can be added to the melt prior to theformation of the fibers. As the resulting fibers cool, the calcium saltcan migrate to the surface of the fiber. In certain embodiments, anamount of calcium salt can be added such that the amount of calcium thatmigrates to the surface of the fiber is sufficient to reduce TSST-1production. The concentration of the calcium salt added to the polymermelt can be any suitable concentration, such as, e.g., between about 10%and about 30%, such as, e.g., between about 15% and about 25% of thefiber weight.

While the distribution of the calcium salt on and/or within an articleof the present invention, such as a tampon, can vary as needed, incertain embodiments, the calcium contained in the one or more portionsof the article can be distributed such that suitable effectiveness forreducing or prohibiting the production of TSST-1 on or within thearticle can be attained. The calcium included in the one or moreportions of an article of the present invention can be fugitive, looselyadhered, bound, partially bound, substantially bound, or any combinationthereof and the like.

An article of the present invention can optionally include otherbeneficial components commonly found in pharmaceutical compositions,such as, for example vitamins, herbs, aloe, moisturizers, botanicals,supplementary antimicrobials, anti-parasitic agents, antipruritics,astringents, local anesthetics, or anti-inflammatory agents. In certainembodiments, the calcium can work in conjunction with one or more of theoptionally included components in a complementary or synergistic way.

The present invention is further illustrated by the following examples,which should not be construed as limiting in any way.

EXAMPLES Example 1

This example demonstrates the reduction of the amount of TSST-1 toxinupon the addition of different calcium salts as measured by the ShakeFlask Method.

Materials and Methods

In this example, the amount of TSST-1 toxin produced by S. aureus wasmeasured using the Shake Flask Method.

The Shake Flask Method was performed in triplicate with appropriatecontrols. Twenty-five ml of Brain Heart Infusion broth (BHI) (Difco) wasdispensed into 250 ml flasks and the flasks were covered. The medium wasautoclaved at 121°-124° C. for 15 minutes and allowed to cool to roomtemperature.

The calcium solution was prepared by dissolving an appropriate amount ofcalcium salt into 2.5 mM Phosphate Buffered Saline (PBS). Dilutions ofcalcium solution were prepared using the following calcium solution (ml)to BHI medium (mls) ratios: 1:49, 5:45, 10:40, and 25:25. Each dilutionwas tested in triplicate. Each dilution was added to a 250 ml ErlenmeyerFlask. The flasks were inoculated with approximately 10⁶ CFU/ml of an18-24 hour culture of Staphylococcus aureus MN 8 then incubated at 37°C. while shaking for 18-20 hours. A corresponding control of PBS and BHIwas also tested. The flasks were removed from the shaker and 3-4 ml offluid was aseptically removed. A standard plate count analysis andEnzyme-Linked ImmunoSorbent Assay (ELISA) were performed using standardtechniques. Results for the test solutions were compared to theappropriate control.

Results

As shown in Table 1, the addition of calcium salt results in a reductionin TSST-1 as measured by the Shake Flask Method. Table 1 furtherdemonstrates that the anion associated with the calcium ion affects theamount of calcium needed to substantially reduce the amount of theTSST-1 toxin measured by the Shake Flask Method.

TABLE 1 Ca (mM) % TSST-1 % TSST-1 Concentration Change Change Calciumadded to (ug/ml) vs. (ug/ml/mM) Salt shake flask control vs. controlchloride 15.1 37 2.5 stearate 9.9 65 6.6 lactate 8.8 49 5.6 citratemalate 6.8 89 13.1

This example demonstrates that, of the calcium salts tested, calciumcitrate malate provides the greatest reduction in TSST-1 toxin at thelowest concentration. Calcium chloride, on the other hand, provides theleast reduction in TSST-1 toxin even with the highest concentration.

Example 2

This example demonstrates the reduction of the amount of TSST-1 toxinupon the addition of calcium salts as measured by the Shake FlaskMethod, along with the solubility of the calcium salts tested.

Materials and Methods

Materials and methods were as described in Example 1.

Results

Table 2 illustrates that the solubility of the calcium salt is relatedto the calcium salt's ability to reduce TSST-1.

TABLE 2 Ca (mM) % TSST-1 % TSST-1 Solubility (mM Concentration ChangeChange (ug/ of Calcium in Calcium added to (ug/ml) vs. ml/mM) vs. H₂O atSalt shake flask control control 25° C. chloride 75.6 82 1.1 7324stearate 98.8 62 0.6 <0.003 lactate 53.1 89 1.7 229 citrate 22.6 88 3.945.1 malate

As shown in Tables 1 and 2, while calcium stearate at low concentrationis more effective than calcium chloride (Table 1) in reducing TSST-1,the effectiveness of calcium stearate does not increase with a higherconcentration due to limited solubility. As materials suitable for usein tampons require a solubility of at least about 0.3 millimoles ofcalcium per L of water to achieve adequate calcium levels in the finaltampon, this example further demonstrates that the solubility of certaincalcium salts that can reduce TSST-1 activity, such as, e.g., calciumstearate, can be too low for effective use in a tampon.

Example 3

This example demonstrates the reduction of the growth of S. aureus andreduction in the amount of TSST-1 toxin upon the addition of calciumsalts as measured by the Shake Flask Method.

Materials and Methods

Materials and methods were as described in Example 1.

Results

Table 3 illustrates that calcium salts can reduce TSST-1 levels asmeasured in the Shake Flask Method, but can also markedly reduce thegrowth of S. aureus relative to the control. Table 3 also shows thatcalcium acetate and calcium ascorbate virtually eliminate the growth ofS. aureus relative to controls at the measured concentration.

TABLE 3 Ca (mM/ml) % TSST-1 Change in Concentration Change S. aureusadded to (ug/ml) vs. CFU/ml vs. Calcium Salt shake flask control controlacetate 62.8 ND >2 log ascorbate 75.2 ND >2 log lactate 88.5 90% <1 logcitrate malate 22.6 88% <1 log

As shown in Table 3, certain calcium salts that reduce TSST-1 productioncan have an undesirable effect on the cell density of S. aureus. Assuch, this example demonstrates that while the addition of certaincalcium salts can reduce TSST-1 levels, the addition of such salts canhave an undesirable effect on the vaginal flora, such as, e.g., makingthose salts unsuitable for use in tampons.

Example 4

This example demonstrates that calcium is more effective than magnesiumfor inhibiting the amount of TSST-1 toxin produced in the Shake FlaskMethod. This example further illustrates that zinc, copper, and ironsalts can be detrimental to levels of S. aureus.

Materials and Methods

Materials and methods were as described in Example 1.

Results

Table 4 shows that calcium is more effective than magnesium in reducingTSST-1 as measured by the Shake Flask Method. Table 4 also illustratesthat while zinc, copper, and iron salts reduce toxin compared tocontrol, such salts do so because they are surprisingly lethal to the S.aureus bacteria when compared to the control.

TABLE 4 Cation (mM) Change in Concentration S. Aureus added to % TSST-1Change CFU/ml vs. Compound shake flask (ug/ml) vs. control controlCalcium chloride 75.6 82% ≦1 log  Magnesium chloride 74.8 47% ≦1 log Zinc chloride 74.8 ND >2 log Copper chloride 74.8 ND >3 log Ferrouschloride 74.8 ND >2 log

As such, this example demonstrates that calcium is more effective thanmagnesium in reducing TSST-1. This example further demonstrates thatcalcium does not negatively impact the growth of S. aureus as does zinc,copper, and/or iron.

Example 5

This example demonstrates that calcium salts have substantially noeffect on Lactobacillus bacteria as measured by the Maximum ToleratedDose Test.

Materials and Methods

In this example, the change in Lactobacillus levels was measured usingthe Maximum Tolerated Dose Test (MTDT). The MTDT was performed asfollows:

Three microorganisms were tested: Lactobacillus crispatus (LMG 12005),Lactobacillus gasseri (ATCC 9857), and Lactobacillus iners (LMG 18916).A macrotube assay using calcium assay solution was performedindividually on each of the three test microorganisms.

L. gasseri was grown for 48 hours under anaerobic conditions inAnaerobic CDM Genital Tract Secretions Media (Anerobe Systems, catalognumber AS-892a). The inoculum was adjusted to approximately 10⁸ CFU/mlby comparison to a McFarland 0.5 standard. The L. crispatus and L. inerswere grown for 48 hours under anaerobic conditions on chocolate agar(Remel) and the inoculum was made in the genital tract secretions mediato a turbidity of approximately 10⁸ CFU/ml by comparison to a McFarland0.5 standard. Plate counts were performed on all test organism inoculumtubes to determine the exact CFU/ml. All organisms were in logarithmicgrowth phase prior to analysis of the assay solutions.

A series of five assay solutions of calcium salts were made. Each tubein the seven tube assay (6 experimental and 1 control) contained 7 ml ofgenital tract secretions media and 3 ml of the stock assay solution orsolvent. To each tube in the series 0.1 ml of each individual testorganism inoculum was added. The assay tubes were incubated at 35° C.under anaerobic conditions. After 48 hours of incubation, a 0.1 mlaliquot of each assay tube solution was diluted in saline (Remel),plated to chocolate agar (Remel), and incubated at 35° C. underanaerobic conditions to determine the number of viable organisms. Platescontaining 30-300 organisms were counted.

Results

Table 5 shows calcium salts can be effective in reducing the TSST-1toxin while having no effect on the keystone Lactobacillus vaginalspecies (L. crispatus, L. gasseri, L. iners) associated with vaginalhealth.

TABLE 5 Ca (mM) Change in Change in Change in Concentration L. cripatusL. gasseri L. iners Calcium Salt added vs. control vs. control vs.control citrate malate 13.5 <1 log <1 log <1 log lacate 53.1 <1 log <1log <1 log

As such, this example and Example 3 demonstrate that calcium salts, suchas, e.g., calcium citrate malate and calcium lactate, can reduce TSST-1while being substantially non-lethal to normal vaginal flora, such as,e.g., lactobacillus.

Example 6

This example demonstrates the moisture gain of various calcium salts asmeasured by the Moisture Gain Test.

Materials and Methods

In this example, the moisture gain of the calcium salts tested wasdetermined by the Moisture Gain Test.

Between 0.500 to 2.000 g of calcium salt was placed into a 20 mlTraceclean vial. Both the tare weight of the empty jar and the jar withthe salt were recorded. The open vial was placed in an oven for 3 hoursat 100° C., and then stored in a desiccator overnight. The jar wassealed and weighed. The “dry weight” of the salt was determined as thedifference between the empty tare weight of the jar with lid and theweight of the jar and lid with the salt following drying and storage inthe desiccator. The sealed jar was placed under test conditions and thelid was immediately removed. At 3.5, 6.5, and 22 hours, the jar wasresealed and weighed. The “wet weight” was determined as the differencebetween the final weight after exposure to 80% RH at 23° C. and the tareweight of the initial jar and lid. The amount of moisture uptake wascalculated as the difference between the “wet weight” and the “dryweight.”

Results

Table 6 illustrates that calcium chloride is deliquescent absorbing over100% of its initial dry weight upon exposure to 80% relative humidity(RH) at 23 degrees C. for 22 hours.

TABLE 6 Moisture Gain by Calcium salts RH80% Calcium chloride Calciumlactate Calcium citrate malate Hours % gain % gain % gain 3.5 20.2 11.914.8 6.5 34.4 17.2 16.7 22 117.4 29.2 17.3

This example demonstrates that certain salts, such as, e.g., calciumchloride, can have high levels of moisture gain that may not be suitableto a tampon. An acceptable level of moisture gain is less than about50%, such as, e.g., less than about 40% as measured by the Moisture Gaintest.

Example 7

This example demonstrates the amount of calcium that can be added to atampon.

Materials and Methods

Calcium solution was added to tampons based on tampon absorbency ascalculated by the Syngyna Test.

Results

Table 7 illustrates the amount of calcium (millimoles) that can be addedto tampons having different absorbency levels.

TABLE 7 Calcium added to Tampon Tampon Absorbency (grams) 4 9 12 14 18Ca (mmoles) 0.009 0.020 0.027 0.034 0.041

This example demonstrates the amount of calcium that can be added to atampon based on the article capacity in certain embodiments.

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “40 mm” is intended to mean“about 40 mm.”

All documents cited in the Detailed Description of the Invention are, inrelevant part, incorporated herein by reference; the citation of anydocument is not to be construed as an admission that it is prior artwith respect to the present invention. To the extent that any meaning ordefinition of a term in this document conflicts with any meaning ordefinition of the same term in a document incorporated by reference, themeaning or definition assigned to that term in this document shallgovern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

What is claimed is:
 1. A method for manufacturing a vaginal article, themethod comprising the steps of: a. forming a tampon pledget comprisingrayon fibers and/or cotton fibers, wherein the rayon fibers and/orcotton fibers include a calcium salt that was added thereto prior toformation of the tampon pledget; and b. compressing and/or shaping thetampon pledget into a finished tampon configuration that is differentthan that of the tampon pledget.
 2. The method of claim 1, wherein thetampon pledget comprises cotton fibers that were washed with a solutionor suspension containing the calcium salt.
 3. The method of claim 1,wherein the tampon pledget comprises rayon fibers and wherein thecalcium salt was included in a fiber finish utilized in the rayon fiberspinning process.
 4. The method of claim 1, wherein a solution orsuspension comprising the calcium salt is sprayed onto the rayon fibersand/or cotton fibers prior to the formation of the tampon pledget. 5.The method of claim 1, wherein the calcium salt is substantiallynon-lethal to Lactobacillus crispatus, Lactobacillus gasseri, andLactobacillus iners when measured by the Maximum Tolerated Dose Testdescribed herein.
 6. The method of claim 1, wherein the calcium saltcomprises calcium lactate and/or calcium citrate malate.
 7. The methodof claim 1, wherein the calcium salt is included in an amount sufficientto reduce the production of TSST-1 by at least 50% when measured by theShake Flask Method described herein.
 8. The method of claim 1, whereinthe calcium salt is included in an amount sufficient to reduce theproduction of TSST-1 by at least 70% when measured by the Shake FlaskMethod described herein.
 9. A method for manufacturing a vaginalarticle, the method comprising the steps of: a. forming a tampon pledgetcomprising rayon, wherein the rayon fibers include a calcium salt thatwas added thereto prior to formation of the tampon pledget; and b.compressing and/or shaping the tampon pledget into a finished tamponconfiguration that is different than that of the tampon pledget, whereinthe calcium salt comprises calcium lactate and/or calcium citratemalate.
 10. The method of claim 9, wherein the calcium salt was includedin a fiber finish utilized in the rayon fiber spinning process.
 11. Themethod of claim 9, wherein a solution or suspension comprising thecalcium salt is sprayed onto the rayon fibers after the rayon fibers areformed in a rayon spinning process.
 12. The method of claim 9, whereinthe calcium salt is included in an amount sufficient to reduce theproduction of TSST-1 by at least 50% when measured by the Shake FlaskMethod described herein.
 13. The method of claim 9, wherein the calciumsalt is included in an amount sufficient to reduce the production ofTSST-1 by at least 70% when measured by the Shake Flask Method describedherein.
 14. The method of claim 9, wherein the calcium salt issubstantially non-lethal to Lactobacillus crispatus, Lactobacillusgasseri, and Lactobacillus iners when measured by the Maximum ToleratedDose Test described herein
 15. A method for manufacturing a vaginalarticle, the method comprising the steps of: a. forming a tampon pledgetcomprising cotton, wherein the cotton fibers include a calcium salt thatwas added thereto prior to formation of the tampon pledget; and b.compressing and/or shaping the tampon pledget into a finished tamponconfiguration that is different than that of the tampon pledget, whereinthe calcium salt comprises calcium lactate and/or calcium citratemalate.
 16. The method of claim 15, wherein the cotton fibers werewashed with a solution or suspension containing the calcium salt. 17.The method of claim 15, wherein a solution or suspension comprising thecalcium salt is sprayed onto the cotton fibers after the cotton fibersare washed.
 18. The method of claim 15, wherein the calcium salt isincluded in an amount sufficient to reduce the production of TSST-1 byat least 50% when measured by the Shake Flask Method described herein.19. The method of claim 15, wherein the calcium salt is included in anamount sufficient to reduce the production of TSST-1 by at least 70%when measured by the Shake Flask Method described herein.
 20. The methodof claim 15, wherein the calcium salt is substantially non-lethal toLactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus inerswhen measured by the Maximum Tolerated Dose Test described herein